Download Decrease in calcitonin and parathyroid hormone mRNA levels and

Histol Histopathol (2001) 16: 407-414
Histology and
Histopathology
http://www.ehu.es/histol-histopathol
Cellular and Molecular Biology
Decrease in calcitonin and parathyroid
hormone mRNA levels and hormone secretion
under long-term hypervitaminosis Dg in rats
J.M. ~ernández-~antosl~*,
J.C. Utrillall*, E. condel, A. Hevia1, M. Loda2 and l. Martín-Lacave1
'Department of Normal and Pathological Citology and Histology, School of Medicine, University of Seville, Spain and 2Department of
Adult Oncology, Dana Farber Cancer Institute, and Department of Pathology, Brighan and Women Hospital, Boston, MA, USA
*Co-first authors
Summary. In calcium homeostasis, vitamin D3 is a
potent serum calcium-raising agent which in vivo
regulates both calcitonin (CT) and parathyroid hormone
(PTH) gene expression. Serum calcium is the major
secretagogue for CT, a h o r m o n s product whose
biosynthesis is the main biological activity of thyroid Ccells. Taking advantage of this regulatory mechanism,
long-term vitamin D3-induced hypercalcemia has been
extensively used as a model to produce hyperactivation,
hyperplasia and even proliferative lesions of C-cells,
supposedly to reduce the sustained high calcium serum
concentrations. We have recently demonstrated that CT
serum levels did not rise after long-term
hypervitaminosis D3. Moreover, C-cells did not have a
proliferative response, rather a decrease in CT-producing
C-cell number was observed. In order to confirm the
inhibitory effect of vitamin D3 on C-cells, Wistar rats
were administered vitamin D3 chronically (25,000 IUId)
with or without calcium chloride (CaC12). Under these
long-term vitaminD -hypercalcemic conditions,
calcium, active metaboetes of vitamin 4 , CT and PTH
serum concentrations were determined by RIA; CT and
PTH mRNA levels were analysed by Northern blot and
in situ hybridization; and, finally, the ultrastructure of
calciotrophic hormone-producing cells was analysed by
electron microscapy. Our results show, that, in rats, long
term administration of vitamin D3 results in a decrease
in hormone biosynthetic activities of both PTH and CTproducing cells, albeit at different magnitudes. Based
upon these results, we conclude that hypervitaminosis
D3-based methods do not stimulate C-cell activity and
can not be used to induce proliferative lesions of
calcitonin-producingcells.
Key words: Vitamin D3, Calcitonin, PTH, C cells,
Parathyroid cells, Hypercalcemia
Orrprint requests to: ln6s Martín-Lacave, Departamento de Citologia e
Histologla Normal y Patológica, Facultad de Medicina, Universidad de
Sevilla, Av. Sanchez Pizjuhn S/N. 41009 Sevilla, Spain. Fax: 34-954551799. e-rnail: [email protected].
lntroduction
Calcitonin (CT), parathyroid hormone (PTH) and
1,25-dihydroxycholecalciferol (1,25-(OH) D3), the
active metabolite of vitamin D3, are the main ormones
involved in regulating calcium homeostasis. CT is
produced mainly in the C cells, which represent about
5% of al1 the endocrine cells in the rat thyroid gland
(Martín-Lacave et al., 1992), while PTH is synthesized
in the chief cells of the parathyroid gland. A high
calcium stimulates CT secretion whereas a low calcium
stimulates PTH secretion (Naveh-Many et al., 1992).
Expression of the PTH gene is regulated by 1,25(OH)2D3 and estrogens (Silver et al., 1985; Naveh-Many
and Silver, 1990; Naveh-Many et al., 1992). PTH
mRNA levels are reduced over 90% 24 hours after 1,25(OH) D3 administration (Silver et al., 1985, 1986;
~ a v e ? i - ~ and
a n ~Silver, 1990). Moreover, 1,25(OH)2D3 is a powerful inhibitor of CT gene
transcription that reduces CT expression to 4% of basa1
within 24 hours (Silver and Naveh-Many, 1993). in
addition, estrogens, as in the case of PTH, induce CT
gene expression. It has also been reported that chronic
hypercalcemia induced by large doses of vitamin D3
produces hypertrophic and hyperplastic changes of Ccells and has even been used as a model to induce
proliferative lesions derived from them (Thurston and
Williams, 1982; Capen and Martin, 1989; Okada et al.,
1991). However, in those studies, plasma calcitonin
levels were not measured to verify the supposed
increasing hormonal synthesis by C-cells.
We have recently demonstrated that during a longterm hypercalcemia induced by vitamin D , calcitonin
serum levels, and, also, the number o calcitonin
immunopositive cells of the thyroid gland, slightly
decreased (Martín-Lacave et al., 1998). Those last
results are in sharp contrast to previous papers reporting
a hypertrophic and hyperplastic effect on C-celis after
vitamin-D treatrnent (Zabel, 1976; Okada et al., 1991).
Nevertheless, the effect of prolonged treatment with
vitamin D3 metabolites on CT and PTH transcription
t
9
Calciotrophic hormones synthesis under hypentitaminosis D3
and secretion by specialized endocrine cells has not been
examined yet. In this study, we present the different
behaviour of C-cells and chief cells in the synthesis and
release of calciotrophic hormones under a long-term
treatment with pharmacological doses of oral vitamin
D3. We demonstrate that vitamin D3-induced hypercaleemia does not stimulate biosynthetic CT activity.
Moreover, vitamin D3 long-term administration leads to
a decrease in CT mRNA and CT serum levels and, also,
in PTH mRNA and seric PTH; results which are not
compatible with hyperactivation-mediated proliferative
changes.
probes, corresponding to 3' and 5' end coding regions,
respectively, was used (Heinrich et al., 1984). For
GAPDH mRNA, a cocktail of two 45-base oligodeoxynucleotide antisense probes corresponding to NH2 and
COOH ends of mature protein, respectively, was used.
Sequences were chosen using the gcg Wisconsin
Package and showed no homology with any other known
sequence of Genebank Database. Antisense probes and
complementary sense chains, used as negative controls,
were 3'-OH end-tailed with Digoxigenin-11-dUTPIdATP,
according to the manufacturer's instruction
(Oligonucleotide Tailing Kit, Roche).
Materials and methods
Northern blof analysis
Experimental model
Male Wistar rats (n=90), aged 2 months, were
divided into groups. Group O (n=10) was the no
treatment control. Group 1 (n=20) was the control
utilized at each timepoint. Group 11 consisted of 20
anirnals receiving 50,000 IU of vitamin D (previously
diluted in an alcoholic vehicle) per 100
of drinking
water for either 1 or 2 months. On average, each subject
drank over 50 m1 of this solution every day. Group 111
consisted of 20 animals receiving the same treatment as
group 11 plus 0.1% of CaC12. Finally, group IV: 20
animals which received only 0.1% CaC12. In the course
of the experiment, the rats were housed and handled
according to approved guidelines, maintained under a
standard laboratory diet and allowed water ad libitum.
Animals were sacrificed under ether anaesthesia. Blood
samples were taken by aortic puncture and thyroparathyroid complexes were extracted and immediately
either fixed or frozen in liquid nitrogen.
Total thyro-parathyroid RNA was isolated as
previously described (Chomczynski and Sacchi, 1987).
Twenty p g of total RNA was size fractionated by
electrophoresis through 1% agarose/2% formaldehyde
gel and transferred to nylon membrane (Bionylon Zt,
Bioprobe). For hybridization, 30 nglml of specific probe
was diluted in SxSSC, 0.1% N-Laurylsarcosyne, 0.02%
SDS, 1% Blocking Reagent (Roche) and incubated
overnight at 6 0 OC. After stringency washes,
digoxigenin-labelled nucleotides were detected with
alkaline phosphatase-linked anti-digoxigenin antibodies
which were finally revealed by quimioluminescence. CT
and PTH hybnd signals were normalized by subsequent
hybridization of membranes with the GAPDH mRNA
specific probe. Analysis of autoradiographs was
performed using an image analysis program (NIH Image
6.01). mRNA levels (the ratio of integrated intensities
CTIGAPDH and PTHIGAPDH) were expressed in
arbitrary units. CT and PTH mRNA levels in treated
groups were expressed as % of control values.
Serum anaiysis
ln situ hybridization
Total serum calcium was determined using a calcium
analyzer (Merck Vitalab Selectra). The leve1 of serum
CT was determined using commercially available kits
(Nichols Institute Diagnostics, CA, USA). The levels of
serum PTH were determined using the Rat PTH (IRMA)
Kit (Nichols Institute Diagnostics, CA, USA). Serum
concentrations of 25-OHD3 were measured by RIA
using a 25-hydroxyvitamin D, 125~RiA Kit (INCSTAR
Corporation-Stillwater, Minnesota, USA). The
measurement of 1,25-(OH)2D3 levels was carried out
using a RIA for the determination of 1,25dihydroxyvitamin D (Nichols Institute Diagnostics CA,
USA). Results were expressed as pg 1,25-(OH)2D3/ml
rat serum.
After the isolation, the thyro-parathyroid complex
was fixed in 4% paraformaldehyde and paraffinembedded. Paraffin-embedded 5 p m sections were
deparaffinized, rehydrated and permeabilised with
Proteinase K (Roche). For hybridization, 2 nglpl of
specific antisense, or control sense oligonucleotide
probe, was diluted in 50% deionized formamide, 10%
dextran sulphate, 2xSSC, 0.lxTris-EDTA, 1 pglpl
sonicated sperm salmon DNA, 5xDenhardt's solution.
Sections were incubated overnight with 40 ,u1 of this
solution, under coverslip, at 42 OC in humidity chamber.
After extensive stringency washes, digoxigenin was
detected using an anti-digoxigenin alkaline phosphataseconjugated antibody which was subsequently detected
by a standard histoenzymatic color reaction. Finally,
sections were methyl green-counterstained and mounted
with glycerol-gelatin.
nd
Probes
For CT mRNA, a 45 base antisense oligodeoxynucleotide probe, complementary to 3' end of exon 4
(Jacobs et al., 1981) was used. For PTH mRNA, a
cocktail of two 45-base oligodeoxynucleotide antisense
Electron micrmcopy
Small fragments of thyroid-parathyroid complexes
Calciotrophic hormones synthesis under hyperviaminosis D3
Table 1. Body weight (g) and total s m m calcium (mgldl) in control and experimental animals.
o
BODY WElGHT (g)
Months
1
339133
371141
347145
369157
480156
360I49*
442142
468160
TREATMENT
Control
V i D3
Vit D3 + CaC12
CaCI,
o
2
SERUM CALCIUM (mgldl)
Months
1
2
538171
353I46W
401143*
398167
MeanISD. *: Pe0.05;m : Pe0.01 *: PeO.001. Statisticai diierences versus control rats of same age.
were fixed in 2.5% glutaradehyde and 2% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, for 4 h,
postfixed in O s o 4 in the same buffer, dehydrated
through graded alcohols, and embedded via propylene
oxide in Spur. Ultrathin sections were counterstained
with uranyl acetate and lead citrate.
Statistical analysis
second main active metabolite of vitamin D3, the 25OHD3: The administration of vitamin D3, combined or
not with CaCl produced an approximately 10-fold
increase of 25-8HD3 serum levels in al1 treated rats, as
compared with control rats, whose basa1 25-OHD3
serum levels did not vary at the daerent time-points of
the experiment (Fig. 1B). In contrast with 25-OHD3
data, the administration of vitamin D3 did not raise the
serum levels of the most active metabolite of vitamin D3
Data were subjected to ANOVA. The mean values in
each experiment were compared with the control group
using Student's test. Results were given as mean ISEM.
P values of less than 0.05 were accepted as significant.
Results
Effects on serum calcium and rat body weight
The administration of vitamin D3 produced a
marked increase in serum calcium concentration as
compared with controls. Calcium concentration one
month after treatment with vitamin D3 raised from 10.54
mg/ml (normocalcemia) to 14.81 mg/ml (Table l), a
state that can be characterized as severe sustained
hypercalcemia. There was no significant change in
calcium levels after two months of treatment. There was
a significant decrease in the weight of the treated rats in
comparison with the control rats. This decrease was
more pronounced in rats without calcium chloride
supplement in the drinking water. These rats also
showed generalized weakness and increasing signs of
cachexia following the treatment. In contrast, animals
treated with calcium chloride done showed stable serum
calcium levels and body weights. Therefore, further
studies were limited to animals treated with vitamin D3.
Effects on serum concentration of vitamin D3 metabolites
The hypervitaminosis D3 state was mainly due to the
Flg. 1. Serum levels of vitamin D3 metabolites in control rats and in rats
treated either with vitamin D3 or vjtamin D3 plus calcium chloride. A. Serum
25-OH-vitamin D3 and 1,25-(OH)2D3levels in control rats. B. Serum 25
OHD3 levels in control and experimental animals. C. Serum 1,25-(OH)2D3
levels in control and experimental animals. Each bar represents the
mean~SEMof 10 rats. *: P ~ 0 . 0 5 ;Pc0.01;
~:
-: P<0.001 versus untreated
rats.
c
rime of tmatment (mnt&bS)
1-
ViL D,
V¡D,+M,
/
Calciotrophic hormones synthesis under hypervitaminosis D3
1,25-(OH)2D3 (Fig. 1C). As a result of the treatment,
decreased serum l,25-(OH)2D3 values were found in al1
experimental animals. This effect was more evident after
the first month of treatment. In relation to this, a
significant decrease in basa1 levels of 1,25-(OHhD3 was
seen in 2-, 3- and 4-month-old control rats, which
diminished more than 60% during this period of their
lives (Fig. 1A).
Table 2. Changes of serum calcitonin and PTH levels under long-ten hypewitaminosis DB
TREATMENT
SERUM CALClTONlN (pgtml)
months
1
2
Control
V I D3
V I D3 + C&I2
21.9412.95
25.16I9.19
29.6017.49
SERUM PTH @g/ml)
months
1
2'
49.3011 0.05
26.7916.30.2813.67-
MeaniSD. *: P<0.05; *: P<0.001. Statistical dierences versus control rats of same age.
A
Time of treahent
1month
2 months
Time of treatment
1 month
2 months
GAPDH mRNA+
1.27 kb
1
Time of trattment (montbs)
Time of treatment (months)
Flg. 2. Detecüon of CT and PTH mRNAs by Northem blot analysis aíter 1 and 2 months of treatment with vitamin D3 and vitamin D, with calcium
chloride. A. Gel blot of total RNA from thyroid-parathyroid tissue hybridized sequentially for CT mRNA and GAPDH mRNA. Twenty pg were applied on
each line. Lanes: 1, control group; 2, vitamin D3-treatedgroup; 3, vitamin D3+CaC12-treatedgroup. Figure represents the results of one representative
out of h e equivalent expeitments. Relative CT mRNA levels of control and experimental rats. Results are shown as the meaniSEM for 5 different
8XperlmentS. B. Gel blot of total RNA from thyroid-parathyroid tissue hybridized sequentially for PTH mRNA and GAPDH mRNA. Twenty pg were
applied on each line. Lanes: 1, control group; 2, vitamin D3-treated group; 3. vitamin D,+CaC12-treated group. Figure represents the results of one
representaüve out of five equhralent experiments. Relative CT mRNA levels of control and experimental rats. Results are shown as the mean~SEMfor
5 diiferent experiments.
Calciotrophic hormones synthesis under hypervitaminosis D3
Effects on CT expression and CT serum concentration
A moderate decrease in the steady-state CT mRNA
levels was found by Northern blot analysis after one
month of vitamin D3 treatment (Fig. 2). This decrease
was fairly small in the group treated with vitamin Dg and
calcium chloride. CT gene expression decreased more
significantly in both groups after two months o f
1-parathyroid gland
treatrnent. A. Control rt
r 2 rnonth
) and PTH mRNAs (DF) by in siiu hybridization on rat i
Fig. 3. Detection of CT
There is a clear localization of CT mRNA in al1 C cells. B. Vltamin D3-treatedrats: There is a reduction in the amount of CT rnRNA which makes the
identiñcation of the C cells d i c u l t in comparison with control glands. C. Vitamin D3+CaC12-treatedrats: the staining of C cells with the CT probe is
more reduced than in control glands but slightly higher in comparison with vitamin D3-treatedrats. x 400. Bar: 25 prn. D. Control rat: There is a clear
localization of W H mRNA in al1 chief cells. E. Vitamin D3-treatedrats: There is a complete reduction in the amount of PTH mRNA which makes the
parathyroidgland negatively stained. F. Vitamin D3+CaC12-treatedrats. P: parathyroid gland; T: thyroid gland. x 200. Bar :50 pm.
'
Calciotrophic hormones synthesis under hypen/itaminosisD3
treatment. Thyroid sections stained by in s i t u
hybridization corroborate the decrease in the amount of
CT mRNA in C cells after vitamin-D treatrnent, however
the proportion of CT mRNA positive cells seemed not to
vary in comparison to controls (Fig. 3). Similarly, the
decrease in serum CT concentration was more
pronounced in the group treated with vitamin D3, and,
also, more evident after the second month of treatment
(Table 2).
Effects on PTH expression and PTH serum
wncentration
The administration of vitamin D3 strongly inhibited
the expression of the PTH gene. As can be seen in Figs.
2,3, the amount of PTH mRNA became undetectable by
Northern blot analysis of 10p g of total thyroparathyroid
RNA and by in situ hybridization. No differences were
obsewed with the addition of calcium chloride. As a
result, PTH levels in serum were markedly decreased
(Table 2), becaming undetectable after one month of
treatment.
Effects on the ultrastructure of C cells and chief cells
In comparison with control rats, secretory granules
of C-cells in al1 treated animals were markedly reduced
in number but they did not disappear completely. The
Golgi apparatus was moderately developed and the
endoplasmic reticulum was mostly vesicular in shape
Flg. 4. Ultrastnictural irnages of Ccells (A, B) and chief cells (C, D) belonging to control rats (A, C) and vitamin D3-treated rats (6, D). As can be
observed, in the thyroid gland (A, B) there is a marked decrease in the nurnber of secretory granules contained in the cytoplasm of the C cells under
hypervitaminosis D,. In the parathyroid gland (C, D), under hype~itamin0SisD, conditions, there is a reduction in the complexity of membrane
infoldings (arrows) in the chief cells, and an increase in the number of empty vesicles (anow heads). x 4,400. Bar: 2,3 prn.
v)
Calciotrophic hormones synthesis under hypervitaminosis D3
and somewhat decreased in volume. Therefore, the
cytoplasm of the degranulated cells was much clearer
than that of the normaliy granulated cells (Fig. 4A,B). In
the parathyroid glands of treated rats, the majority of
chief cells were characterized by the presence of
numerous empty vesicles of different sizes in the
cytoplasm (Fig. 4C,D). Furthermore, the experimental
rats presented chief cells with less complex infoldings of
the cellular membrane than in control animals. Different
organelles such as rough endoplasmic reticulum and free
ribosomes, appeared to be similar to those of non-treated
rats.
Discussion
The results of our investigations, together with some
literature data, support our first hypothesis that C-cells,
in a hypercalcemic state due to an intoxication with
vitamin D3, do not respond with hypertrophic and
hyperplastic changes, but rather in these conditions, the
main biosynthetic activity of C-cells is inhibited
(Martín-Lacave et al., 1998). The hypervitaminosis D3
state obtained in our experimental model was mainly due
to the rising of 25-OHD3 serum levels. 25-OHD3 is the
first active and most abundant metabolite in the pathway
of vitamin D3 activation (Parfitt, 1999). Conversion of
vitamin D3 in 25-OHD is not subjected to strict
regulation and basical y depends on vitamin D3
availability (Reichel et al., 1989). In our experiment,
non-treated experimental rats, aged 2, 3 and 4 months,
showed similar serum 25-OHD3 values, whereas the oral
administration of vitamin D3 led to a ten-fold increased
in serum levels of 25-OHD . In contrast, synthesis and
degradation of 1,25-(OH)
(the most active vitamin
D3 metabolite) are strict y down regulated by serum
calcium, and up regulated by calcitonin and PTH
(Kawashirna et al., 1981; Shigematsu et al., 1986; Sinki
et al., 1992). Based on this, as a result of the
hypercalcemia and decreased levels of serum calcitonin
and PTH, lower levels of 1,25-(OH)2D3 were observed
in both treatment groups. In our study we have also
obsewed a decrease in 1,25-(0 D3 serum levels with
age in control animals. A similar ecrease has also been
described in humans with aging (Fujisawa et al., 1984;
Quesada et al., 1994). This observation, in our
experimental model, might be related to developmental
calcium requirements rather than changes caused by the
prolongation of the treatment.
In chronic hypervitaminosis D , such as in the
experimental model we have presented, parathyroid cells
must respond to both Ca++ and vitamin D3: which act
synergistically on PTH expression and secretion. Steadystate levels of PTH mRNA dramaticaly decreased (as
assessed by either Northern blotting or in situ
hybridization). This, in turn, results in undetectable
levels of serum PTH. These patterns of PTH expression
and secretion are similar to those elicited by short-term
treatments with 1,25-(OH)2D3 (Karmali et al., 1989;
Naveh-Many et al., 1989, 1992; Naveh-Many and Silver
1990; Moallem et al., 1998). Ultrastructural appearance
?
Td3
9
of the parathyroid gland in long-term vitamin-treated
rats also coincides with that seen in chief cells under
short-term hypercalcemia or hypervitaminosis D
(Setoguti et al., 1981, 1995; Wild et al., 1985). The
apparent degradation of storage grandes by hydrolysis,
and the decrease of the cell surface area, seems to be
closely associated with an intracellular regulatory
mechanism for PTH secretion.
HypeMtaminosis D3 afEects CT-producing celís in a
distinctly different manner. C-cell population may
respond to the increase in extracellular calcium by
releasing CT stored in granules through exocytosis.
However, vitamin Dgmreduces the transcription of the CT
gene. CT secretion @ves the cells an almost completely
degranulated appearance under electron microscopy.
Nonetheless, they stiIl conserve morphological signs of
the persistence of some hormonal synthesis. These
ultrastructural findings concur with those obtaiited by in
situ hybridization and Northern blot analysis. Based
upon these, C-cells, still have a moderate rate of CT
synthesis which is not observable in short-term
treatments, where CT mRNA decreased 4% of baseline
level at 48 h Waveh-Many and Silver, 1988; Silver and
Naveh-Many, 1993). Importantly, in these studies, 1,25(OH)2D3 did not raise serum calcium. These results
indicate that in the case of C-celis, where hyperdcemia
and hypervitaminosis DJ are antagonic stimuli, a second
hormone synthesis regulatory mechanism, besides the
vitamin D3-mediated transcriptional inhibition or mRNA
stability, would be present.
In the group treated with vitamin D? and CaCi2, CT
mRNA levels were higher than those elicited by vitamin
D3 alone. In this case, the effect of calcium ingestion on
the release of different mediators at the gastrointestinal
level, such as gastrin, cholecystokinin and glucagon,
might be a decisive factor. These hormones are wellknown CT secretagogues and are also involved in
regulating calcitonin gene tranwiption (Naueh-Many
and Silver, 1988; Raue et al., 1991, 1993). The
administration of CaC12 with vitamin D3 in the drinking
water may thus act as a "buf€erl'against the 25-OHD3 induced blockade of CT synthesis.
In numerous studies in which vitamin D3 has been
used as a serurn-calcium-raising agent, to investigate the
reaction of C cells to long-term hypercalcemia, C-cell
hypertrophy and hyperplasia has been described (Zabel,
1976; Thurston and Williams, 1982; Kameda et al.,
1985; Capen and Martin, 1989; Okada et al., 1991). We
have recently described (Martln-Lacave et al., 1998) the
effect of long-term administration of vitamin D3 at
hyperdcemic doses on the thyroid gland of the rat. In
contrast bo previous reports, we have found that C cells
did not respond with hypertrophic and hyperplastic
changes. In addition, the number of C cells that were
immunopositive for CT decreased. This observation
concurs with the findings of our current study in which
vitamin D does not stimulate the C-cell population, but
rather inhhits CT synthesis. Based upon these resulb it
is difficult to accep-t that hypewitaminosis D ,which
inhibits the main biosynthetic activity of c-ceds, could
Calciotrophic hormones synthesis under hypen/itaminosisD3
induce proliferative changes in this endocrine
population. We conclude that the oral administration of
vitamin D3 at serum-calcium-raising doses for prolonged
periods reduces the rate of biosynthesis and secretion of
both PTH and CT.Therefore, supporting our previous
data (Martín-Lacave et al., 1998), hypervitaminosis D3based methods are not reliable models to induce
proliferative lesions of calcitonin-producing cells, as has
been extensively described in the literature.
Acknowledgments. This work was supported by the National Fund for
Medical Research of the Social Security (F.I.S.S.S.), Spain (0571).
Capen C.C. and Martin S.L. (1989). The effects of xenobiotics on the
structure and functlon of thyroid follicular and C-cells. Toxicol.
Pathol. 17,266-293.
Chomczynski P. and Sacchi N. (1987). Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162, 156-159.
Fujisawa Y., Kaichi K. and Matsuda H. (1984). Role of change in
Vitamin D metabolism with age in calcium and phosphorus
metabolism in normal human subjects. J. Clin. Endocrinol. Metab.
59,719-726.
Jacobs J.W., Chin W.W., Dee P.C., Habener J.F., Goodman R.H., Bell
N.H. and Potts J.T. (1981). Calcitonin messenger RNA encodes
multiple polypeptides in a single precursor. Science 213, 457-459.
Kameda Y., Ito M., Ogawa K. and Tagawa T. (1985). Alterations of
immunoreactive somatostatin in thyroid C cells after induced
hypercalcemia, hypocalcemia, and antithyroid drug treatment. Anat
Rec. 211,34-42.
Karmali R., Farrow S., Hewison M., Barker S. and O'Riordan J.L.H.
(1989). Effects of 1,25-dihydroxyvitarnin D3 and corüsol on bobine
and human parathyroidcells. J. Endocrinol. 123, 137-142.
Kawashima H., Torikai S. and Kurokawa R. (1981). Calcitonin
selectively stimulates 25-hydrxyvitamin D3-1hydroxilase in the
pmximal straight tubules of rat kidney. Nature 298,327-329.
Heinrich G., Kronenberg H.M., Poots J.T. and Habener J.F. (1984).
Gene encoding parathyroid hormone. J. Biol. Chem. 259, 33203329.
Marlln-Lacave l., Conde E., Montero C. and Galera-Davidson H. (1992).
Quantitative changes in the Frequency and distribution of the C-cell
population in the rat thyroid gland with age. Cell Tissue Res. 270,
73-77.
Martín-Lacave l.,Ramos F., Uhilla J.C., Conde E., Hevia A., Fernández
R., Moreno A.M., Fernández-Santos J.M. and Galera-Davidson H.
(1998). Chronic hypewitaminosis D3 determines a decrease in C
cells number and calcitonin levels in rats. J. Endocrinol. lnvest 21,
102-108.
Moallem E., Kllav R., Silver J. and Naveh-Many T. (1988). RNA-Protein
binding and post-transcriptional regulation of parathyroid hormone
gene expression by calcium and phosphate J. Biol. Chem. 273,
5253-5259.
Naveh-Many T. and Silver J. (1988). Regulation of calcitonin gene
transcription by vitamin D metabolites in vivo in the rat. J. Clin.
Invest. 81,270-273.
Naveh-Many T. and Silver J. (1990). RegulaUon of parathyroid Hormone
Gene Expresion by hipocalcemia, Hypercalcemia, and Vitamin D in
the rat. J. Clin. Invest. 86, 1313-1319.
Naveh-Many T., Almogi G., Livni N. and Silver J. (1992). Estrogen
receptors and biologic response in rat parathyroid ttissue and Ccells. J. Clin. Invest. 90,2434-2438.
Naveh-Many T., Friedlaender M.M., Mayer H. and Silver J. (1989).
Calcium regulates parathyroid hormone messenger ribonucleic acid
(mRNA), but not calcitonin mRNA in vivo in the rat. Dominant role of
1,25-dihydroxyvitamin D. Endocrinology 125,275-280.
Okada H., Toyota N,, Harimaya Y. and Matsuzawa K. (1991). Immunohistochemical alterations of C cells in sheep treated with vitamin D.
J. Comp. Pathol. 105,263-270.
Parfitt K. (1999). Manrtindale. The complete drug referente.
Pharmaceutical Press. Taunton, Massachussetts. pp 1366-1369.
Quesada J.M., Mateo A,, Jans l., Rodriguez M. and Bouillon R. (1994).
Calcitriol corrects deficient calcitonin secretion in the vitamin Ddeficient elderly. J. Bone Min. Res. 9, 53-57.
Raue F., Zink A. and Scherübl H. (1991). Regulation of calcitonin
secretion. In: Medullary thyroid carcinoma. Calmettes C. and
Guliana J.M. (eds). Colloque INSERMJJohn Cibbey Eurotest Ltd. pp
3138.
Raue F., Zink A. and Scherübl H. (1993). Regulation of calcitonin
secretionin vitro. Horm. Metab. Res. 25,473-476.
Reichel H., KoefRer P. and Norman AW. (1989). The role of the vitamin
D endocrine system in health and disease. N. Engl. J. Med. 320,
980-991.
Setoguti T., lnoue Y. and Kato K. (1981). Electron-microscopic studies
on the relationship between the frequency of parathyroid storage
granules and serum calcium levels in the rat. Cell Tissue Res. 219,
457-467.
Setoguti S., lnoue Y. and Wild P. (1995). The biological significance of
storage granules in rat parathyroid cells. Microsc. Res. Tech. 32,
148-163.
Shigematsu T., Horiuchi N,, Ogura Y., Myahara T. and Suda T. (1986).
Human parathyroid hormone inhibits renal 24-hydroxylase activity of
25-hydroxyvitamin D3 by a mechanism involving adenosine 3', 5'monophosphatein rats. Endocrinology 118, 1583-1589.
Silver J. and Naveh-Many T. (1993). Calcitonin gene regulation in vivo.
Horm. Metab. Res. 25,470-472.
Silver J., Naveh-Many T., Mayer H., Schmelzer J. and Popovher M.M.
(1986). Regulation by vitamin D metabolites of parathyroid hormone
gene transcriptionin vivo in the rat. J. Clin. Invest. 78, 1296-1301.
Silver J., Russell J. and Sherwood L.M. (1985). Regulation by vitamin D
metabolites of messenger ribonucleic acid for preproparathyroid
hormone in isolated bovine parathyroid cells. Proc. Natl. Acad. Sci.
USA 82,4270-4273.
Sinki T., Jin C.H., Nishimura A., Nagai Y., Ohyama Y., Noshiro M.,
Okuda K. and Suda T. (1992). Parathyroid hormone inhibits 25hydroxy-vitaminD3-24-hydroxylase mRNA expression stimulated by
l,25-hydroxyntamin Dj in rat kidney but not intestine. J. Biol. Chem.
267, 13757-13762.
Thurston V. and Williams E.D. (1982). Experimental induction of C cell
tumours in thyroid by increased dietary content of vitamin 4. Acta
Endocrinol. 100, 41-45.
Wild P., Gloor S. and Vetsch E. (1985). Quantitative aspects of
membrane behavior in rat parathyroid cells after depression or
elevation of serum calcium. Lab. Invest. 52,490-495.
Zabel M: (1976). The response of thyroid C cell system in rat to longterm hypercalcemia. Endocrinology 67,315-321.
Accepted November 16,2000